Human cells have a variety of receptors on their surfaces. Inter alia, G-protein coupled receptors (hereinafter referred to as “GPCR” or “GPCRs”) comprise one of the largest protein families of transmembrane receptors. The human genome retains approximately 30,000 human genes, as many as 1000 of which are known to encode GPCRs. On the basis of recent studies conducted on vertebrate genomes, GPCRs have been grouped into five classes. The first class comprises a rhodopsin receptor family to which 670 receptor proteins belong. This rhodopsin receptor family can react with various ligands including amines (alpha group), peptides (beta group), lipid-like substances (gamma group), nucleotides, and glycoproteins (delta group), and comprises a lot of drug target receptors. The second class addresses the secretin receptor family and has binding domains for peptide hormones. Receptors in this family are associated with homeostasis and have been arising as important targets for drug development. The third class is assigned the adhesion receptor family, characterized by a GPCR proteolytic site (GPS). The development of drugs targeting GPCR members of this family has not yet taken place because they exhibit various N-terminal moieties and little is known about their ligands. Within the fourth class is the glutamate receptor family in which 22 GPCR members have so far been identified. Relatively little is known about the specificity of each protein. The last class is the Frizzled/Taste2 family that encompasses 10 Frizzled receptors for which Wnt glycoproteins serve as ligands, 5 SMO (smoothened) receptors which need no ligands, and 25 Taste2 receptors which are required for sensing various tastes. Receptors including GPCRs are also classified on the basis of the identification of endogenous ligands. Receptors bind with known endogenous compounds or are classified as orphan receptors whose endogenous ligands have not yet been identified.
GPCRs are found in a broad range of types of tissues and cells and are associated with many different physiological mechanisms. They are activated by a wide range of ligands, for example, hormones such as thyroid-stimulating hormone (TSH), adrenocorticotropic hormone, glucagon and vasopressin, amines such as 5-HT, acetylcholine (muscarinic AchR), and histamines, lipids such as LPA and S1P, and signal transmitters such as amino acids, Ca2+, nucleic acids, peptides and light. The wide distribution and diversity of roles that GPCRs play is evidence to the important roles that they play in various pathological diseases. Indeed, GPCRs are known to be involved in various diseases including bronchoconstriction, hypertension, diabetes, inflammation, hormone disorders, cell death, cancer, neurotransmission and behavioral disorders. Currently, GPCRs are therefore an area that is important to the development of pharmaceutical products. Approximately 360 GPCRs are now considered available for drug development. Of these, 46 have already been used for drug development while the remaining about 320 genes can be exploited for drug development. There are approximately an estimated 150 Orphan GPCRs (oGPCRs). In the field of new drug development, cell membrane receptors act as selective sites for drug action and are responsible for 50% of all drug targets (Nature Reviews Drug Discovery, 2004. 2008) and GPCR activity modulating drugs, inter alia, account for 30% of the most frequently used top 100 drugs (40 billion dollars, 9% of the total drug market). Therefore, GPCR is one of the most significant targets for the development of new drugs (Nature Reviews Drug Discovery, 2004, 2008).
GPCRs have common structural features. All of these receptors have seven hydrophobic membrane-spanning domains, each 20˜30 amino acids long, which are connected by hydrophilic amino acid sequences of various lengths. The receptors have an extracellular N-terminus while the C-terminus is located in the cytoplasm. GTP-binding proteins (G proteins) act as mediators transmitting to intracellular effectors the signals that are generated by binding hormones or other chemical ligands that stimulate GPCR. After a ligand has become bound to GPCR, the intracellular domains of the receptor undergo a conformational change which allows the receptor to interact with G protein, which in turn activates intracellular signal transmitters such as adenylate cyclase, phospholipase C or ion channel. This system generates a signaling cascade in which many secondary transmitters act in response to the binding of one ligand to GPCR. This mechanism is used by cells to detect extracellular environmental changes and to properly react in response to the changes. On the whole, receptors are activated by endogenous ligands with the concomitant generation of a conformational change, which allows association between the receptors and the G-proteins. Recent studies on the interaction between proteins have revealed that GPCR is associated with various proteins such as GRK or SH2 domain (src homology 2 domain)-containing proteins, and adaptor Grb2 as well as G protein to participate in signaling transduction.
Under normal conditions, signaling transduction brings about the final result which is cell activation or suppression. In a physiological environment, GPCRs exist in equilibrium between their inactive and active states in the cell membrane. Inactive receptors cannot exert a biological response in conjunction with cellular signal transduction pathways. The receptors can exhibit biological responses via a signal transduction pathway (through G-proteins) only when they have structurally changed to their active form. The receptor may be stabilized into an active form by compounds such as endogenous ligands or drugs. Therefore, functional studies, such as the cloning of such gene families, and the identification of new ligands thereof, have the same meaning as the development of new drug candidates, that is, siRNA, antibodies, polypeptides, effectors, inhibitors, agonists, antagonists, etc.
Development, differentiation, homeostasis, responses to stimuli, control of the cell cycle, as well as the aging and apoptosis of living organisms are mostly a result of the selective expression of specific genes within cells. This is true for cellular mechanisms associated with diseases. Particularly, pathological phenomena, such as oncogenesis, are induced by gene mutations that in the end lead to changes in gene expression.
According to various studies into oncogenesis, the generation of tumors is the result of the accumulation of various genetic changes such as the loss of chromosomal heterozygosity, the activation of oncogenes, the inactivation of tumor suppressor genes including p53 gene, etc. (Bishop, J. M., Cell, 64:249-270 (1991)). Further, the activation of oncogenes by oncogene amplification was reported to account for 10-30% of cancer cases. Thus, the activation of oncogenes is significant to the pathological study of various cancers. There is an imminent need for identifying oncogenes and developing a method of controlling the oncogenes.